1. Field of the Invention
The present invention relates to a novel enzyme, sorbitol oxidase, a process for producing the same, and the use thereof.
2. Description of the Related Art
Up to now, sorbitol oxidase, a novel enzyme, which catalyzes a reaction in which D-sorbitol is oxidized to D-glucose and hydrogen peroxide has not been found. Sorbitol is a compound which is produced in vivo from glucose by aldose reductase. Because sorbitol has poor membrane permeability, the sorbitol has a tendency to be accumulated in a cell such as a red blood cell. The accumulation of sorbitol causes functional disorders such as the increase in an osmotic pressure in vivo, the swelling of a cell, etc. Each of the functional disorders is considered as one of the diabetic complications. Thus, the content of sorbitol in a red blood cell derived from a body can be used for the diagnosis of diabetic complications.
Recently, a number of blocking agents for aldose reductase have been developed as therapeutic agents for diabetic complications. For confirming the effects of these therapeutic agents, the measurement of sorbitol in a red blood cell has been the subject of serious studies paid much attention to.
A sorbitol dehydrogenase (L-Iditol: NAD.sup.+ 2-oxidoreductase (EC. 1.1.1.14)), which catalyzes the following reaction, has been used as a method for determining D-sorbitol. EQU D-sorbitol+NAD.sup.+ .revreaction.D-fructose+NADH+H.sup.+
According to the above method, a sorbitol dehydrogenase is allowed to react with D-sorbitol in a biological sample to produce NADH. Then, the NADH thus produced is measured, thereby determining the content of D-sorbitol in the biological sample. However, this method has the following problems.
Since the molecular extinction coefficient of NADH is small, the sensitivity of the measurement is low. In addition, for measuring the NADH with good sensitivity, a special apparatus is required. That is, the NADH is measured with good sensitivity by fluorometry at a wavelength of 366 nm (excitation) and 452 nm (emission). Moreover, NADH, NAD.sup.+, etc., which are included in the biological sample may hinder the sensitivity of the measurement.
Another method for determining D-sorbitol, using the above-mentioned sorbitol dehydrogenase has been utilized, in which an artificial electron receptor is used. This method is more useful than the above-mentioned method for measuring NADH; however, the sensitivity for the measurement in this method is not sufficiently precise for clinical diagnosis. Further, sorbitol dehydrogenase is a membrane-bound enzyme, so that it is unstable and tends to be inactivated.
In order to overcome the above-mentioned problems involved in the determination of D-sorbitol, attempts to develop another enzyme such as a sorbitol oxidase have been tried, but have not been found.